Antibacterial Activity of an FtsZ Inhibitor Celastrol and Its Synergistic Effect with Vancomycin against Enterococci In Vitro and In Vivo

ABSTRACT Enterococci can cause various infectious diseases, including urinary tract infection, wound infection, and life-threatening endocarditis and meningitis. The emergence and transmission of vancomycin-resistant enterococci (VRE) have presented a challenge to clinical treatment. There is an urgent need to develop new strategies to fight against this pathogen. This study investigated the antibacterial and anti-biofilm activity of celastrol (CEL), a natural product originating from Tripterygium wilfordii Hook F, against enterococci, and its adjuvant capacity of restoring the susceptibility of VRE to vancomycin in vitro and in vivo. CEL inhibited all enterococcus strains tested, with MICs ranging from 0.5 to 4 μg/mL. More than 50% of biofilm was eliminated by CEL at 16 μg/mL after 24 h of exposure. The combination of CEL and vancomycin showed a synergistic effect against all 23 strains tested in checkerboard assays. The combination of sub-MIC levels of CEL and vancomycin showed a synergistic effect in a time-kill assay and exhibited significant protective efficacy in Galleria mellonella larval infection model compared with either drug used alone. The underlying mechanisms of CEL were explored by conducting biomolecular binding interactions and an enzyme inhibition assay of CEL on bacterial cell-division protein FtsZ. CEL presented strong binding and suppression ability to FtsZ, with Kd and IC50 values of 2.454 μM and 1.04 ± 0.17 μg/mL, respectively. CEL exhibits a significant antibacterial and synergic activity against VRE in vitro and in vivo and has the potential to be a new antibacterial agent or adjuvant to vancomycin as a therapeutic option in combating VRE. IMPORTANCE The emergence and transmission of VRE pose a significant medical and public health challenge. CEL, well-known for a wide range of biological activities, has not previously been investigated for its synergistic effect with vancomycin against VRE. In the present study, CEL exhibited antibacterial activity against enterococci, including VRE strains, and restored the activity of vancomycin against VRE in vitro and in vivo. Hence, CEL has the potential to be a new antibacterial adjuvant to vancomycin and could provide a promising therapeutic option in combating VRE.

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The manuscript by Lu et al. examines the synergistic effect of celastrol with vancomycin against Enterococcus faecalis and Enterococcus faecium. Enterococci are serious health concern and the emergence of vancomycin resistant strains highlights the need for new treatment strategies. The research presented here shows that celastrol (CEL) displays anti-biofilm activity and synergizes with vancomycin to kill Enterococci including vancomycin-resistant strains (VRE) in vitro. Additionally, using a Galleria melonella larvae infection model, Liu et al. show that the combination of CEL and vancomycin protected larvae from lethal infection of VRE compared to either compound alone. Lui et al. observed elongated cells in SEM micrographs and hypothesized that the filamenting temperature-sensitive mutant Z (FtsZ) might be the target of CEL. They demonstrated by surface plasmon resonance analysis the CEL does have a strong binding ability with FtsZ and suppressed the GTPase activity that is essential for regulating cell division. Overall, the work presented here is very solid. A major highlight is the exciting finding that CEL can render VRE strains sensitive to vancomycin both in-vitro and in-vivo.
Minor comments: 1. The impact of the work shown is somewhat diminished by the fact that the same phenomena reported here has previously been reported in S. aureus.
2. In figure legend 2 the strains and strain numbers are listed. It would be helpful if the authors could indicate which strains are VRE in the figure legend so the reader does not have to refer back to Table 1. 3. The authors showed that the combination of CLE and vancomycin protect from lethal infection in a G. melonella model. It would be interesting to know if this combination protects in a mammalian model system.
4. While perhaps difficult, it would be nice if the authors could quantify the differences in cell length.
5. The authors assert that CEL targets FtsZ and this allows for synergy with vancomycin. Do other known inhibitors of FtsZ also promote synergy with vancomycin? Does ftsZ mutation in VRE confer susceptibility to vancomycin? 6. One drawback is the correlation with CEL binding FtsZ and suppressing GTPase activity as the primary mechanism of action in inducing vancomycin susceptibility. Would CEL still synergize with a VRE strain harboring a GTPase deficient FtsZ?

Dr. Conlon
Editor, Microbiology Spectrum, Re: Manuscript Spectrum03699-22 entitled "Antibacterial activity of celastrol and its synergistic effect with vancomycin against enterococci in vitro and in vivo" Dear Dr. Conlon, Thank you and the reviewers for the thoughtful and careful consideration of our manuscript. We appreciate the favorable review and potential acceptability of the revised paper. We have responded to the comments in detail and revised the manuscript accordingly (highlighted with yellow color). We believe that the current revision addresses all the critique points. Below is a point-by-point response to both reviewers.

Reviewer #1
1. While I found the experimental results compelling, the writing could be significantly improved. This would not only help readability but also the impact that these exciting results will have with their audience.

Response:
Thanks for pointing this out. We got help from Carol Mita Ann of Harvard Medical School for revision, went over the manuscript and made changes where necessary.

In general, the use of color in the Figures 2-4 would help the reader.
Response: Thanks for your suggestion. We made changes accordingly and found it's very helpful to make it easy for the readers to understand the figures by using color.
The changed figures as follows:  3. Related to the "time-kill" assay, I would like to note that VAN is generally cited as having a bacteriostatic effect on enterococci and the mechanism of FtsZ inhibition is also suggestive to slow/impair division rather than have an active mechanism of killing (such as membrane disruption). Moreover, the time-dependence of the CFU reduction and the fact that in all cases a substantial number of bacterial cells were recovered after 24 h, the use of the term bacteriocidial may be an overstatement.
There does not appear to be a rapid mode of action but rather, I anticipate, a multifactorial process that ultimately leads to cell death. While the authors define bacteriocidal as a reduction by 3log10 units ( Response: Thanks for the suggestion, and we agree with the editor. All of the six strains are VRE, and the corresponding change has been made in the revised manuscript, please see Line 503-504 in the revised version.

The authors showed that the combination of CLE and vancomycin protect from lethal infection in a G. melonella model. It would be interesting to know if this combination protects in a mammalian model system.
Response: Thanks for the suggestion. We did the pilot study of the in vivo efficacy of CEL on a mouse infection model not long ago, with 3 mice per group. Mice were infected peritoneally with 1x10 7 CFU cells of E. faecalis strain ATCC 700802. Groups of three mice were given vancomycin (4 mg/kg), CEL (4 mg/kg), or combination of vancomycin and CEL 2h post infection. The mice were sacrificed and CFU recovered from mouse organs including hearts, livers, spleens, lungs and kidneys were numerated. The results are as follows: It looked promising. We are considering to do further study in the future to optimize the doses of bacterial inocula and the dose of CEL and vancomycin used in vivo.
Besides, more mice will be used in each group to get reliable results.

While perhaps difficult, it would be nice if the authors could quantify the differences in cell length.
Response: As suggested, the estimated cell length of the bacteria after the treatment of CEL or not was added. Please see Lines 149-153 and 155-156 in the revised version. FtsZ is a cell division essential gene conserved in most bacteria, therefore, it is difficult to construct or select the mutant strains. To the best of our knowledge, the reported ftsZ mutations in gram positive bacteria were all screened resistant mutants at concentrations higher than MIC. We tried this assay, however no mutant survived.

The authors assert that CEL targets
Instead, the ftsZ-knockdown Bacillus subtilis (B. subtilis) strain by CRISPR interference (CRISPRi) from B. subtilis CRISPRi Essential Gene Knockdown Library was employed in the present study. Gene knockdown was induced by xylose (1%, freshly prepared) in the CRISPRi strain. The no sgRNA wild type strain, an unrelated gene ispH knockdown strain, and the uninduced ftsZ CRSPRi strain (no xylose added) were used as controls. As shown in Table 3  Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. You will find the reviewers' comments below. Specifically, reviewer 1 recommends integrating the new in vitro data displayed in the response to reviewers document.
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Brian Conlon
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): I thank the authors for their responses to my suggestions and concerns; however, I am puzzled as to the reason for omission of newly obtained data from the manuscript. In response to comment 3, the authors performed the requested experiment but did not update Fig 4. While I understand the reluctance to include the preliminary mouse study, the in vitro experiment showing additional concentrations/combinations of CEL and VAN could be easily included in the manuscript. In fact, this was requested for the broader audience. I am recommending that it is integrated into Figure 4 -perhaps just the higher concentrations inset into Fig 4A -or that it is provided to readers as a supplemental figure.
Minor comment The image resolution of Figure 7 (right hand side) and 8 are low.
Reviewer #2 (Comments for the Author): The authors have adequately addressed the previous reviewer comments.